Use of Leptospira fainei serovar hurstbridge bacteria for diagnosing Leptospirosis

ABSTRACT

The present invention discloses an in vitro method for diagnosing a  Leptospira  infection in a biological sample of a subject, comprising a step of contacting said sample with bacterial cells of a serovar of the  Leptospira fainei  species, preferably bacterial cells of the  Leptospira fainei  serovar Hurstbridge, or an antigenic fraction of said bacterial cells. In a preferred embodiment, said  Leptospira  infection is not due to bacteria belonging to the serovar of the  Leptospira fainei  species used in the diagnostic method.

BACKGROUND OF THE INVENTION

Outbreaks of leptospirosis are of significant and growing public healthconcern in many tropical and subtropical countries. Leptospirosis is azoonotic disease caused by spirochaetes of the genus Leptospira, whichare classified into 9 pathogenic species and more than 200 serovars onthe basis of structural heterogeneity in the carbohydrate component ofthe lipopolysaccharide. Human infections are endemic in most tropicaland most moderate climates. Globally, an estimated number of >500,000severe cases occur annually with fatality rates exceeding 10%.

This neglected infectious disease is also reported to be an emerging orre-emerging disease in industrialized countries, with probableincreasing impacts due to global warming and increasing travel-relatedcases [Lau C, Travel Med Infect Dis. 2010]. The incidence isunderestimated due to highly variable clinical presentation which ischaracterized by non-specific signs and symptoms; leptospirosis is oftenconfused with other diseases such as dengue, rickettsiosis, entericfevers and malaria. The complete triad of Weil's disease (hepaticfailure, renal failure and hemorrhage) is recognized to account for lessthan one third of human cases [Mac Bride A. J. et al., Curr. Opin.Infect. Dis. 2005]. Most of the early signs and symptoms point to theso-called “acute fever of unknown origin” (FUO), a major diagnosticchallenge in tropical and subtropical areas. Because of nonspecificsymptoms in human leptospirosis, the biological confirmation is neededto ascertain the disease. In many endemic regions, the laboratorydiagnosis of leptospirosis is not available due to a lack of reliable,rapid and simple diagnostic assay for a point of care diagnosis of humanleptospirosis.

Yet, an early and proper antibiotic treatment is a key determinant ofthe outcome in leptospirosis [Suputtamongkol Y. et al, PLoSNegl. Trop.Dis. 2010], because, in contrast to many similar diseases (e.g. dengue),leptospirosis can easily be treated with antibiotics in its earlystages. However, the diagnosis has to be confirmed before the 5th dayafter disease onset, when treatment with antibiotics is most effective.

The need for reliable Rapid Diagnostic Tests (RDTs) for diagnosing humanleptospirosis has therefore been largely recognized. Preferably, theseRDTs should be portable, so that they can be used directly on thebedside, even in remote health centers, so as to improve clinicalmanagement of leptospirosis patients in remote dispensaries of tropicaland subtropical regions.

Currently, the laboratory diagnosis for leptospirosis relies on thedetection of antibodies raised against the Leptospira bacteria byserological techniques such as Microscopic agglutination test (MAT), orof spirochaete nucleic acids by PCR [Goarant C. Trop. Med. Int. Health,2009). However, these techniques are inappropriate for early clinicalcare in peripheral health centers that support the major part of theleptospirosis burden, because they are time-consuming and requiresophisticated materials that are most frequently available only incentral reference laboratories [Hartskeerl R A, Clin. Microbiol. Infect.2011]. Also, serological techniques such as Microscopic agglutinationtest (MAT) are limited by the fact that they use few representativegroup of serovar antigens, so that a negative reaction on serial samplesdoes not rule out the possibility that the patient might actually beinfected with a Leptospira serovar not included in the battery of thetested antigens. PCR-based techniques, although very sensitive and earlytests, are technically demanding and the leptospires disappear from theblood vessels approximately at day 7 after infection.

Moreover, ELISA-based assays using crude whole-cell lysates ofLeptospira strains (usually the saprophyte L. biflexa serovar Patocstrain Patoc 1) as antigens may not recognize the diversity ofcirculating strains and the sensitivity of these tests is generally poor(Mc Bride A J. et al, Curr. Opinion Infect Dis, 2005).

Thus, the biological confirmation of leptospirosis is currently notsatisfactory, as it is not reliable, tedious and rarely available in atimely manner.

In fact, a major challenge is still to discover antigens that areconserved across the major Leptospira strains, since such antigens wouldpotentially be recognized by most of the antibodies generated inLeptospira-infected patients.

In this context, the present inventors have identified a Leptospiraantigen which allows the detection of a broad spectrum of antibodiesdirected against most of the serovars. This Leptospira antigen isexpressed by the bacteria of the serovar Hurstbridge, which wasoriginally isolated from pigs in Australia [Perolat P. et al, Int. J.Syst. Bacteriol. 1998] and is now classified in the species Leptospirafainei, known to also infect humans [Chappel R J. et al, Epidemiol.Infect., 1998]. The present inventors indeed demonstrated that thisantigen presents a high reactivity towards antibodies generated byseveral leptospirosis serogroups, even distantly serologically related,such as serogroups Australis, Autumnalis, Ballum, Bataviae, Canicola,Cynopteri, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Panama,Pomona, Pyrogenes, Sejroe and Tarassovi, on serum samples of a number ofmetropolitan French patients. Of note, false positive results areexcluded since the serovar Hurstbridge is poorly represented outsideAustralia and New Zealand. The present inventors also identified amethod to inactivate the bacteria of the serovar Hurstbridge so as toenhance the exposure of said antigen at the bacterial cell surface.

Using such an antigen, it is possible to develop different RapidDiagnostic Tests (RDTs) exhibiting high sensitivity and specificity fornumerous Leptospira serovars.

As disclosed below in detail, the present inventors developed twodifferent RDTs, that are i) an ELISA test and ii) a vertical flowimmunochromatography dipstick assay, these two RDTs usingheat-inactivated Leptospira fainei bacteria as antigen, for detectinganti-Leptospira human IgM in human serum samples. The robustness ofthese two RDTs is very satisfactory in terms of sensitivity,specificity, reproducibility, and shelf-life.

It is possible to use the ELISA and dipstick assay of the invention asrobust, simple, and rapid diagnostic tools for diagnosing leptospirosisin patients presenting early signs and symptoms thereof.

Moreover, as the dipstick assay of the invention does not requireexpensive and complex analysis system, it can therefore be used inremote health centers, or in remote dispensaries of tropical andsubtropical regions.

As Leptospira fainei bacteria presents a high reactivity towardsantibodies generated by several leptospirosis serogroups, the presentinventors also propose to use Leptospira fainei bacteria as antigen in aMicroscopic Agglutination Test (MAT).

FIGURE LEGENDS

FIG. 1 discloses the Positive and Negative Predictive Values (PPV, NPV)for the diagnosis of leptospirosis using the IgM dipstick of theinvention, using 187 positive and 221 negative serum specimens.

FIG. 2 discloses comparative Positive and Negative Predictive values(PPV, NPV) for the diagnosis of leptospirosis using the IgM dipstick ofthe invention and a commercial lateral flow IgM assay. This comparisonwas conducted on 72 positive and 72 negative serum specimens randomlyselected from New Caledonian specimens.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention discloses an in vitro methodfor diagnosing a Leptospira infection in a biological sample of asubject, comprising a step of contacting said sample with bacterialcells of a serovar of the Leptospira fainei species, or an antigenicfraction of said bacterial cells.

In this aspect, the said Leptospira infection is not due to bacteriabelonging to the said serovar. In other words, when the diagnosticmethod of the invention uses bacteria of a define serovar of theLeptospira fainei species, then this method is not intended to be usedfor diagnosing an infection mediated by this define serovar.

As used herein, the term “antigen” herein means any molecule (e.g.,protein, lipoprotein, polysaccharide, and/or glycoprotein) that causesthe immune system to produce antibodies against the said substance. An“immunogenic” antigen is a specific type of antigen which is able tostimulate an adaptive immune response if injected on its own. At themolecular level, an antigen is thus characterized by its ability to berecognized and “bound” by the antigen-binding site of an antibody.

In the context of the present invention, the term “antigenic fractionthe invention” designates an antigen which is expressed specifically bybacterial cells of the Leptospira fainei species. This antigen isaccessible especially when said bacteria have been inactivated with aheating and optionally with a chemical treatment. Importantly, thisantigen is expressed by a number of bacteria of other serogroups orserovars. More precisely, the antigenic fraction of the inventioncorresponds to an antigen i) which is expressed by the bacterial cellsof the Leptospira fainei species and ii) which is recognized byantibodies that are found in the serum of subjects infected withbacteria belonging to the following serogroups: Australis, Autumnalis,Ballum, Bataviae, Canicola, Cynopteri, Grippotyphosa, Hebdomadis,Icterohaemorrhagiae, Panama, Pomona, Pyrogenes, Sejroe and Tarassovi.

L. fainei belongs to the intermediate group of Leptospira [Perolat P.,Int. J. Syst. Bacteriol., 1998]. Without being bound by the theory, itis hypothesized that this species may share common antigenic featureswith saprophytes and pathogens which constitute the two otherphylogenetic groups in the genus Leptospira. This would explain why theantigen of the invention is recognized by several anti-Leptospiraantibodies, and in particular antibodies generated in subjects which areinfected with bacteria that do not belong to the L. fainei species.

The antigenic fraction of the invention can be a protein, a lipoprotein,a polysaccharide, and/or a glycoprotein. It is preferablylipopolysaccharide (LPS, which remains intact upon heating of thebacterial cells), a secreted protein, a cytoplasmic protein, or aprotein of the cellular membrane of the bacteria of the Leptospirafainei serovar Hurstbridge, for example an envelope membrane protein.More preferably, the said antigenic fraction is LPS.

In the context of the present invention, an antigenic fraction (or anantigen) is said to be “recognized”, “specifically recognized” or“bound” by an antibody if said antibody has an affinity constant K_(a)(which is the inverted dissociation constant, i.e. 1/K_(d)) higher than10⁵ M⁻¹, preferably higher than 10⁶ M⁻¹, more preferably higher than 10⁷M⁻¹ for said antigen (or epitope).

In a preferred embodiment, the diagnosis method of the inventionrequires the use of bacterial cells (or antigenic fraction thereof) ofthe Leptospira fainei serovar Hurstbridge. In a preferred embodiment,the said bacteria are cells of the strain BUT 6, which were originallyisolated from pigs in Australia [Perolat P. et al, Int. J. Syst.Bacteriol., 1998] and are now classified in the species Leptospirafainei [Chappel R J. et al, Epidemiol. Infect., 1998]. These bacteriahave been referenced as L. fainei Hurstbridge 1 and are available ininternational collections (e.g. ATCC BAA-1109^(T) or CRBIP6.1209^(T)).The 16S rDNA partial sequence of the prototype strain of L. fainei isreferenced as GeneBank accession no. U60594, AY631885, AY995712 andFJ154578. The diagnosis method of the invention preferably usesbacterial cells of the Leptospira fainei species that have beenpreviously inactivated or killed, preferably by a heating treatment.Some of these treatments are described in Terpstra W J. et al.,Zentralbl Bakteriol A. 1980.

These particular treatments are referred to hereafter as “inactivatingmethods” of bacterial cells. They enable to inactivate bacterial cellsof the Leptospira fainei species so as to induce the appearance of theantigen of the invention, which can then be used in the diagnosis methodof the invention.

In one embodiment, whole bacterial cells of the Leptospira faineispecies are used. These whole bacterial cells are obtained preferably byinactivating them (preferably by heating, as described below), and/or bytreating them with conservative agents. These conservative agents arewell-known in the art.

In one embodiment, the bacterial cells of the Leptospira fainei speciesare inactivated by means of the so-called “inactivating method of theinvention”, which comprises at least a step of inactivating the saidbacterial cells with a mechanical treatment (e.g., by means of a FrenchPress, sonication, etc.) or a heating treatment (e.g., at a hightemperature). This inactivating method of the invention is preferablyperformed on bacterial cells of the Leptospira fainei serovarHurstridge.

The inactivating method of the invention preferably comprises at least aheating treatment. Thus, the inactivated bacterial cells used in theinvention are preferably heat-inactivated. In this heat-basedinactivating method, the heating step is typically performed bysubmitting the live bacterial cells of the Leptospira fainei species tohigh temperature for at least about 10 minutes, preferably for at leastabout 20 minutes, and more preferably for at least about 30 minutes, andat most for about 1 hour. In order to inactivate efficiently (i.e., atleast 90% of the bacterial cells and preferably almost 100% of them) thetemperature is preferably comprised between about 60° C. and 150° C.,more preferably between about 90° C. and 110° C. and even morepreferably around 100° C. This heating treatment can be performed in anysystem providing a high temperature during the required time (e.g., inan oven, a dry heater, or a boiling bath). Preferably, the heatingtreatment is performed in a boiling bath. In this case, it is preferablethat the cells are kept isolated from the water in a safe recipient.

In another embodiment, the heat- or mechanically-inactivated bacterialcells of the Leptospira fainei species are further treated chemically,for example with an inactivating chemical agent.

In this embodiment, said inactivating chemical agent is defined as anychemical agent having preservation and/or antiseptic properties. Inparticular, preservation of the morphology (shape and structure) of thebacterial cells is important in the context of the invention, as theantigen of the invention may be expressed at the surface of thebacterial cells. Consequently, said inactivating chemical agent ispreferably a fixative agent, for example chosen in the group consistingof: aldehyde (such as formaldehyde, glutaraldehyde, andparaformaldehyde), alcohols (such as methanol, ethanol), acetone,oxidizing agents (such as osmium tetroxide, potassium dichromate,chromic acid, and potassium permanganate) and the like. Saidinactivating chemical agent is more preferably chosen in the groupconsisting of: formaldehyde, and paraformaldehyde. The chemicaltreatment should last sufficiently so that at least 95% of the bacterialcells (and preferably almost 100% of them) are inactivated (in otherwords, no living bacterial cells should remain in the bacteria sample).The chemical treatment will last typically at least about one hour, andmore preferably at least about two hours, and even more than 10 hours,maximally about 24 hours. The duration and the temperature of thereaction obviously depend on the choice of the chemical agent and on itsconcentration. Since chemical agents which can be used for inactivatingbacteria have been known and commercialized for decades, it will be easyfor the skilled person to determine the conditions (e.g., duration,temperature, concentration, etc.) to use for inactivating at least 95%of (and preferably all) the bacterial cells with any given chemicalagent. For example, as disclosed below, the diagnosis method of theinvention requires the use of bacterial cells that have been treatedwith a 0.2% v/v of formaldehyde (formalin) for two to six hours.

Importantly, the mechanical, the heating or the chemical inactivatingtreatment can be performed in any order.

However, in a preferred embodiment, the bacterial cells of theLeptospira fainei species are first treated chemically with aninactivating chemical agent as defined above, and are secondly heated ata high temperature or mechanically, for example in the conditionsdefined previously. In a more preferred embodiment, the diagnosis methodof the invention requires the use of bacterial cells of the Leptospirafainei species that have been first treated with, as an inactivatingchemical treatment, 0.2% v/v of formaldehyde (formalin) during two tosix hours, and then been heated during 45 minutes in a boiling bath.

As used herein, the term “biological sample” refers to any sample thatmay contain Leptospira-specific antibodies, such as any biological fluidfor example serum sample, plasma sample, blood sample, lymph sample,urine sample, or sample of cerebrospinal fluid (CSF). Blood plasma isthe liquid component of blood, in which the blood cells are suspended.It is composed of mostly water (90% by volume), and contains dissolvedproteins, glucose, clotting factors, mineral ions, hormones and carbondioxide. It is easily recovered as it corresponds to the supernatantfluid obtained when non-coagulated blood has been centrifuged. To obtainplasma samples, the blood is mixed with an appropriate amount ofanticoagulant like heparin, oxalate or ethylene diamine tetra-aceticacid (EDTA), then mixed immediately and thoroughly to avoid clotting.Blood serum corresponds to blood plasma without fibrinogen or the otherclotting factors. These proteins are sometimes considered as interferingsubstances in some tests since they can react with the reagent andthereby yield inaccurate results. Serum is therefore often preferred inclinical testing. Serum samples can be easily obtained by the skilledperson by centrifuging blood samples at about 2500 rpm for a few minutesand then recovering the supernatant.

In a preferred embodiment, the diagnosis method of the invention isperformed on a biological sample which is a serum sample. Such a serumsample is obtained by a completely harmless blood collection from thesubject and thus allows for a non-invasive diagnosis of leptospirosis.

In the context of the present invention, the term “subject” hereindesignates human or animal individuals.

The method of the invention is usually performed on subjects who aresuspected of a leptospirosis. The said subjects usually present at leastone symptom known to be associated with such an infection. This symptomis for example fever, chills, myalgia, and/or intense headache.

The method of the invention can also be applied to any individual havingsuffered from leptospirosis, in order to detect the serotype prevalenceof anti-leptospirosis antibodies during or after leptospirosis epidemicevent.

In the context of the invention, the expression “diagnosing a Leptospirainfection” refers to the process of identifying or detecting if asubject or a patient is infected or not with pathogenic bacteria of thegenus Leptospira. It also refers to the process of monitoring theprogression of an infection by said bacteria. In other terms, the methodof the invention enables to diagnose a Leptospira-induced infection in asubject. It also allows discriminating subjects infected by Leptospirabacteria from subjects infected by bacteria from other genus.

It is reminded here that, when the antigen used in the method of theinvention is bacterial cells of a defined serovar of the Leptospirafainei species, the Leptospira infection which is diagnosed according tothe method of the invention is not necessarily due to (or caused by)bacteria belonging to the said serovar. In particular, when the antigenused in the method of the invention is bacterial cells of the Leptospirafainei serovar Hurstbridge, the Leptospira infection which is diagnosedaccording to the method of the invention is not necessarily due to (orcaused by) bacteria belonging to the Leptospira fainei serovarHurstbridge. In a preferred embodiment, the Leptospira infection whichis diagnosed according to the method of the invention is not due to (orcaused by) any bacteria belonging to the Leptospira fainei species, and,in particular, to bacteria belonging to the Leptospira fainei serovarHurstbridge. In a more preferred embodiment, the leptospirosis infectionwhich is diagnosed according to the method of the invention is ratherdue to (or caused by) bacteria belonging to one or more serogroup(s)chosen from Hardjo, Louisiana, Shermani, Celledoni, Sarmin, Javanica,Grippotyphosa, Mini, Serjoe, Australis, Autumnalis, Ballum, Bataviae,Canicola, Cynopteri, Hebdomadis, Icterohaemorrhagiae, Panama, Pomona,Pyrogenes, Sejroe or Tarassovi. In an even more preferred embodiment,the leptospirosis infection which is diagnosed according to the methodof the invention is due to (or caused by) bacteria belonging to one ormore serogroup(s) chosen from Australis, Autumnalis, Ballum, Bataviae,Canicola, Cynopteri, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae,Panama, Pomona, Pyrogenes, Sejroe and Tarassovi.

As disclosed herein, the term “in vitro” refers to studies orexperiments that are conducted using biological samples (e.g. blood orserum samples) which have been isolated from their host organisms (e.g.animals or humans). These experiments can be for example reduced topractice in laboratory materials such as tubes, flasks, wells,microtubes, etc. In contrast, when used herein, the term “in vivo”refers to studies that are conducted on whole living organisms.

An antibody (Ab) is a large Y-shaped protein produced by B-cells that isused by the immune system to identify and neutralize foreign objectssuch as bacteria and viruses. Native antibodies are usuallyheterotetrameric glycoproteins of about 150,000 daltons, comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain comprises a heavy chain variableregion (or domain) (abbreviated herein as HCVR or VH) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, CH1, CH2 and CH3. Each light chain comprises a light chainvariable region (abbreviated herein as LCVR or VL) and a light chainconstant region. The light chain constant region comprises one domain,CL. The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (CDR) or“hypervariable regions”, which are primarily responsible for binding anepitope of an antigen, and which are interspersed with regions that aremore conserved, termed framework regions (FR). Each VH and VL iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.effector cells) and the first component (Clq) of the classicalcomplement system. Constant regions are not involved directly in bindingan antibody to an antigen, but exhibit various effector functions.Depending on the amino acid sequence of the constant region of theirheavy chains, antibodies or immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. Of thevarious human immunoglobulin classes, only human IgG1, IgG2, IgG3 andIgM are known to activate complement; and human IgG1 and IgG3 mediateADCC more efficiently than IgG2 and IgG4.

Antibodies of IgM and IgG isotypes are composed of two identical heavychains and two identical light chains joined by disulfide bonds.Importantly, IgM antibodies form polymers where multiple immunoglobulinsare covalently linked together through disulfide bonds, mostly as apentamer but also as a hexamer. In their pentamer form, they have amolecular mass of approximately 900 kDa. Because each monomer has twoantigen binding sites, a pentameric IgM has 10 binding sites. IgMantibodies cannot bind 10 antigens at the same time because of stericconstraints. Due to its polymeric nature, IgM possesses high avidity.

Of note, IgM antibodies are the first to appear in response to initialexposure to antigen, and the presence of specific IgG, in general,corresponds to maturation of the antibody response.

With the diagnosis assays of the prior art, both IgG and IgM typeantibodies were usually detected in the biological sample of thepatients. In contrast, however, the dipstick and ELISA assays of theinvention are specific to IgM type antibodies induced by the Leptospirainfection, so that the leptospirosis is diagnosed at an early stage andcan therefore be more efficiently cured. In addition, and because IgMtiters are known to decline faster than IgG, some positive MAT resultsmay reveal IgGs remaining from previous exposure to leptospires, andcould therefore be less specific than IgM-specific assays to detectacute and recent leptospirosis.

The identification of the IgM antibodies is performed by using eitherpolyclonal antibodies, or monoclonal antibodies, or antibody functionalfragments, used as “revealing agent”. As used herein, “antibodyfunctional fragments” is intended to include Fab, Fab′, F(ab′)2, scFv,dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof andbispecific antibody fragments. Antibodies can be fragmented usingconventional techniques. For example, F(ab′)2 fragments can be generatedby treating the antibody with pepsin. The resulting F(ab′)2 fragment canbe treated to reduce disulfide bridges to produce Fab′ fragments. Papaindigestion can lead to the formation of Fab fragments. Fab, Fab′ andF(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques. A “monoclonal antibody”, as used herein, meansan antibody arising from a nearly homogeneous antibody population. Moreparticularly, the individual antibodies of a population are identicalexcept for a few possible naturally-occurring mutations which can befound in minimal proportions. In other words, a monoclonal antibodyconsists of a homogeneous antibody arising from the growth of a singlecell clone (for example a hybridoma, a eukaryotic host cell transfectedwith a DNA molecule coding for the homogeneous antibody, a prokaryotichost cell transfected with a DNA molecule coding for the homogeneousantibody, etc.) and is generally characterized by heavy chains of oneand only one class and subclass, and light chains of only one type.Monoclonal antibodies are highly specific and are directed against asingle antigen. In addition, in contrast with preparations of polyclonalantibodies which typically include various antibodies directed againstvarious determinants, or epitopes, each monoclonal antibody is directedagainst a single epitope of the antigen.

Of note, the diagnosis method of the invention can use either livebacterial cells or inactivated bacterial cells.

Live bacterial cells of the Leptospira fainei species can be used forexample in a MAT assay. Such a MAT assay consists in serial dilutions ofthe patient's serum kept in contact with an equal volume of a well grownsuspension of leptospires and read microscopically by estimating 50%agglutination as the end-point titer of the reaction mixture. This assayis broadly used in the art. Briefly, the bacterial cells are grown in anappropriate medium (typically the dedicated Ellinghausen and Mc Culloughmodified Johnson & Harris (EMJH) medium) and put in contact withdifferent dilutions of the tested serum. The agglutination is thenobserved under a dark field microscope.

Advantageously, although it is performed with bacteria of a definedserovar (for example Hurtsbridge), the MAT assay of the inventionenables to diagnose an infection due to bacteria belonging to otherserogroups and/or serovars as mentioned above.

In a preferred embodiment, however, the diagnosis method of theinvention uses bacterial cells of the Leptospira fainei species whichhave been inactivated according to the “inactivating method” of theinvention (as defined above) or antigenic fractions thereof.

In this case, the diagnosis method of the invention can be either anELISA assay, or a dipstick assay. In these assays of the invention, theheat- and optionally chemically-inactivated bacterial cells of theLeptospira fainei species are immobilized on a solid support. Saidsupport is preferably a nitrocellulose membrane for the dipstick assay,or a microtiter plate for the ELISA assay. Alternative supports that canbe used in these assays are well-known by the skilled person.

As used herein, the “ELISA assay of the invention” designates an ELISA(Enzyme-Linked Immunosorbent Assay) assay requiring the use of bacterialcells of the Leptospira fainei species that have been inactivatedaccording to the inactivating method of the invention, or antigenicfractions thereof, said bacteria or fractions being immobilized on asolid support, preferably a microtiter plate. An ELISA assay accordingto the invention is disclosed in the experimental part of theapplication. ELISA assays are broadly used and well-described in theart.

As used herein, the “dipstick assay of the invention” designates adisptick assay requiring the use of bacterial cells of the Leptospirafainei species that have been inactivated according to the inactivatingmethod of the invention, or antigenic fractions thereof, said bacteriaor fractions being immobilized on a solid support, preferably anitrocellulose membrane. A dipstick assay according to the invention isdisclosed in the experimental part of the application. Dipstick assaysare broadly used and well-described in the art.

Interestingly, in the present study, 11 out of 17 confirmedleptospirosis patients could be diagnosed earlier with the Dipstickassay of the invention than with the MAT (see Table 2 below). Similarly,in 16 out of 99 early sera from confirmed cases, the Dipstick assay ofthe invention was positive before the seroascension (10/37) orseroconversion (6/62) could be evidenced with the MAT. Therefore, theIgM dipstick assay of the invention presents an earlier positivity whencompared to MAT in serial sera. Because an early diagnosis is of primeimportance in the clinical management of leptospirosis, the possibilityto ascertain the disease earlier in the course of the infection shouldbe regarded as a real asset.

Immobilization of the bacteria (or fractions thereof) on the solidsupport is performed by conventional means which are well-known in theart. In case of a dipstick assay, the bacteria or fractions thereof canbe for example sprayed on the solid support and let dry until totalevaporation of the sprayed bacteria or fractions thereof. In case of anELISA assay, the bacteria or antigenic fractions thereof can be put incontact with the surface of the solid support and let dry until totalevaporation, for example during at least 6 hours at about 4° C., thenthe unbound bacteria (or fractions) are washed out by successivewashings.

The solid support on which the bacteria (or antigenic fractions) areimmobilized is then put in contact with the biological sample of thesubject, in appropriate conditions so that the human immunoglobulinsthat are present in the said biological sample can bind to theimmobilized bacteria (or fractions thereof). The said appropriateconditions (temperature, duration, etc.) depend on each kind of support,and it is routine matter to identify them for the bacteria (orfractions) of the invention. For example, few seconds (maximally onehour) are sufficient for the dipstick strip of the invention, while afew minutes (maximally twelve hours) are required for the ELISA assay ofthe invention.

The diagnostic method of the invention, and in particular the ELISA anddipstick assays of the invention, further comprises a step of detectingthe human immunoglobulins bound to the immobilized antigen. Thisdetection is typically performed with a revealing agent. This “revealingagent” can be an antibody or a functional fragment of same, either inthe form of an immunoconjugate, or a labeled antibody in order to obtaina detectable and/or quantifiable signal.

In a preferred embodiment, said revealing agent is preferably ananti-human IgM antibody, more preferably an anti-human IgM antibodywhich is conjugated with a detectable label. Examples of detectablelabels include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorot[pi]azinylamine fluorescein, dansylchloride or phycoerythrin; example of luminescent material includesluminol, and examples of bioluminescent materials include luciferase,luciferin, and aequorin; examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

It is also possible to use the antigen of the invention coupled withbeads or nanoparticules. Coated beads or nanoparticles carrying theantigen of the invention would aggregate or immunocaptured in thepresence of a subject's serum containing antibodies recognizing thisantigen. This aggregation is easily detectable by conventional means,such as by microscopy, by flow cytometer, or naked eyes, etc.

The diagnostic assays of the invention advantageously contain a positivecontrol to which the revealing agent binds without requiring thepresence of the antigen-human immunoglobulin complexes. For example, ifthe revealing agent is anti-human IgM antibody, the positive control canconsist in human IgM or at least the constant part thereof.

When the detection step reveals that the biological sample containshuman immunoglobulins that are specific to the antigen of the invention,the said subject is diagnosed to have a Leptospira infection. It is thenpossible to administer an appropriate antibiotic treatment.

Antibiotic treatments that are currently used to prevent or fightagainst leptospirosis are well known to the skilled artisan. Suchantibiotics include doxycycline, ceftriaxone, penicillin G andpenicillin A. They are reviewed in Levett P. N. et al, Mandell, Douglas,and Bennett's Principales and Practice of Infectious Diseases, 2010.

Accordingly, in a second aspect, the present invention provides a methodfor identifying in vitro if a subject will benefit from the treatmentwith an antibiotic-containing composition, the method comprising:

-   -   a) conducting the diagnosis method of the invention on a        biological sample of said subject, as defined above, using        bacterial cells of a serovar of the Leptospira fainei species or        an antigenic fraction of said bacterial cells, and    -   b) identifying if the subject will benefit from the treatment        with an antibiotic-containing composition if the said biological        sample contains human immunoglobulins that are specific to the        said bacterial cells of the Leptospira fainei species, or to        said antigenic fraction of said bacterial cells.

Preferably, the bacterial cells used in this method are bacterial cellsof the Leptospira fainei species that have been inactivated according tothe inactivating method of the invention described above.

Preferably, the bacterial cells used in this method belong to theLeptospira fainei serovar Hurstbridge.

More preferably, the bacterial cells used in this method are bacterialcells of the Leptospira fainei serovar Hurstbridge that have beeninactivated according to the inactivating method of the invention.

As meant herein, a subject is “predicted to benefit from the treatmentwith an antibiotic-containing composition” if it is diagnosed to sufferfrom leptospirosis by the diagnosis method of the invention. This methodis a very useful tool to avoid exposing antibiotics to subjects that donot suffer from leptospirosis, thereby preventing the emergence ofbacterial resistance.

In another aspect, the present invention is drawn to anantibiotic-containing composition for use in treating a subject who hasbeen diagnosed with leptospirosis using the method of the invention.

The present invention also relates to the use of an antibiotic forpreparing a composition intended to treat a subject who has beendiagnosed with leptospirosis using the diagnosis method of theinvention.

Moreover, the present invention related to a method of treating asubject in need thereof, the method comprising administering anantibiotic-containing composition in subjects whose leptospirosis hasbeen diagnosed using the diagnosis method of the invention.

In a second aspect, the present disclosure also provides kits useful forcarrying out the diagnosis method described above.

The kits of the invention generally comprise a solid support coated withinactivated bacterial cells of the Leptospira fainei species, orantigenic fractions of said bacterial cells, and a revealing agent.

In a preferred embodiment, the said revealing agent is a labeledantibody, more preferably a labeled anti-human IgM antibody. Examples ofdetectable labels include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorot[pi]azinylamine fluorescein, dansylchloride or phycoerythrin; example of luminescent material includesluminol, and examples of bioluminescent materials include luciferase,luciferin, and aequorin; examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In a preferred embodiment, the bacterial cells coated on the said solidsupport belong to the Leptospira fainei serovar Hurstbridge. In a morepreferred embodiment, the said bacterial cells are bacterial cells ofthe Leptospira fainei serovar Hurstbridge that have been inactivatedwith the inactivating method of the invention.

In a preferred embodiment, the solid support of said kit is a microtiterplate or a nitrocellulose membrane.

In a preferred embodiment, the kit of the invention further comprises acontrol sample which is also recognized by said revealing agent. Forexample, if the revealing agent is an anti-human IgM antibody, thepositive control can consist in human IgM or at least the constant partthereof.

The kits of the invention can also include instructions for interpretingthe results obtained using the kit.

Kits may also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kits can further comprise componentsnecessary for detecting the detectable label (e.g., an enzyme or asubstrate). Each component of a kit can be enclosed within an individualcontainer and all of the various containers can be within a singlepackage, along with instructions for interpreting the results of theassays performed using the kit.

In another aspect, the present invention targets bacterial cells of theLeptospira fainei species, that have been inactivated with theinactivating method of the invention, or antigenic fractions of saidbacterial cells. As mentioned previously, these bacterial cells orantigenic fractions thereof comprise at least one universal antigen thatis recognized by a number of antibodies specific for Leptospira bacteriabelonging to different serovars and/or serogroups.

Preferably, these bacterial cells belong to the Leptospira faineiserovar Hurstbridge.

In a preferred embodiment, said antigenic fraction is chosen from:liposaccharide, cytoplasmic proteins, secreted proteins, and envelopmembrane proteins. It is more preferably liposaccharide (LPS).

In another aspect, the present invention is drawn to the use of:

i) bacterial cells of the Leptospira fainei species that have beeninactivated with the inactivating method of the invention, or antigenicfractions of said bacterial cells, or

ii) the kit of the invention, containing said bacterial cells and arevealing agent, in a diagnostic method for detecting leptospirosisinfection in a subject in need thereof.

In a preferred embodiment, the said bacterial cells are bacterial cellsof the Leptospira fainei serovar Hurstbridge, that have been preferablyinactivated with the inactivating method of the invention.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

EXAMPLES

I. Material and Methods

1. Antigen Preparation

The antigen was prepared at Institut Pasteur, Unite de Biologie desSpirochetes, Paris, France. A 10 mL EMJH preculture of Leptospira faineiHurstbridge BUT 6^(T) was used to inoculate one liter EMJH. Thisone-liter culture was incubated at 30° C. for 4-7 days with constantshaking until reaching an Optical Density greater or equal to 0.5 at 420nm. The culture was then left standing at room temperature for 2-6 hoursafter addition of 2 mL of 37% (i.e. 0.2% v/v) of formaldehyde (orformalin). The formalin-killed culture was heated for 45 minutes in aboiling water bath. Lastly, the pH was adjusted to 9.6 and thispreparation was stored at 4° C. This crude preparation was directly usedas a fixed antigen in the RDTs (ELISA and dipsticks).

2. Development and Production of the ELISA Assay

2.1. Serum Samples

Blood samples from healthy volunteers with no history of leptospirosisand seronegative by MAT were collected from biobanque (PlatformICAReB/Investigation Clinique et Accès aux Ressources Biologiques,Institut Pasteur):

Serum samples negative and positive for leptospirosis were addressed tothe National Reference Center of Leptospirosis (Institut Pasteur) fordiagnostic purpose between 2010 and 2011 and originated from patientsfrom mainland France and the French West Indies (Guadeloupe andMartinique).

The following groups were established based on the MAT (MicroscopicAgglutination Test) which is the reference test for the serodiagnosis ofleptospirosis so far (gold standard):

-   -   Group A (Negative controls): Two serologies for the same patient        and whose results remained negative in MAT    -   Group B (Positive cases): Two serologies for the same patient        with a serocoversion or seroconversion at demonstrated (>=4-fold        rise) in MAT with at least one pathogenic Leptospira serogroup        and was therefore considered as a laboratory-confirmed        leptospirosis case.        2.2. ELISA Protocol

Flat-bottom microtiter plates (Immulon 1B Thermo, Dutscher) were coatedwith 75 μL of the well-homogenized antigenic solution, overnight at 4°C. Alternatively the antigenic solution was left to evaporate at 37° C.for 1-3 days. The coated plates were then stored in a dry place at roomtemperature up to two years.

Plates were washed three times with PBS-0.2% Tween 20 (PBST), thenincubated for 1 h at 37° C. (or overnight at 4° C.) with blockingsolution, 75 μL PBS-Milk 5% (PBSM)(powders:Dutscher) pH 7.2. Plates werewashed again three times with PBST. Duplicates of 75 μL dilution of thepatient's sera in PBSM (1:400) were incubated for 1 h at 37° C. Onedilution range of a pool of positive serum samples (1:400 to 1:204 800)and one negative control were included on each plate as internalstandards. This pool was constituted by 50 positive serum samplesexhibiting MAT reciprocal titers 800 with at least one pathogenicLeptospira serogroup.

The plates were washed three times with PBST. 75 μL of rabbit anti-humanIgM-peroxidase conjugate (Biorad) diluted at 1:500 in PBSM were added toeach well and plates were incubated for 1 h at 37° C.

Plates were washed five times with PBST. 75 μL of substrate buffer (0.5mM 2-2′-azino di-ethyl benz-thiazoline-6-sulphonic acid (ABTS) (Roche),were added to each well. Plates were read at 415 nm with ELISA reader(Biorad) after 30 min of incubation in the dark at 37° C.

The end-point cut-off was established by titration as the mean OD 405value of the 20 serum samples negative for leptospirosis plus 3 standarddeviations.

2.3. Results of the ELISA Protocol

A total of 449 sera were analyzed:

Technique MAT ELISA IgM negative 308 (TN) 304 positive 141(TP) 135 TN =Truth Negative = 308 TP = Truth Positive = 141 FP = False-Positive = 4FN = False-Negative = 6Sensitivity:TP/(TP+FN)×100=141/(141+6)×100=96%Specificity:TN/(TN+FP)×100=308/(308+4)=99%Positive Predictive Value (VPP):TP/(TP+FP)×100=141/(141+4)×100=97%Negative Predictive Value (VPN):TN/(TN+FN)×100=308/(308+6)×100=98%Precocity of the Test:

MAT−/ MAT−/ MAT+/ n = 282(141patients) Elisa− Elisa+ Elisa− MAT+/Elisa+first serologies n = 80 n = 60 n = 1 n = 0 (57%) (42.5%) (0.5%) secondserologies n = 0 n = 0 n = 6 n = 135(95.7%) (4.3%)

In more than half of the cases (57%), the ELISA assay of the inventionwas positive on the first serum, whereas the MAT was negative.

3. Development and Production of the Dipstick Assay

3.1. Serum Samples

Leptospirosis cases were defined as confirmed when a clinical andepidemiological suspicion was complemented by either a positive specificPCR evidencing genomes of pathogenic Leptospira sp. in the blood orurine of the patient or when two serological analyses using thereference MAT on acute and convalescent sera showed a seroconversion(from nil to a reciprocal titer 400) or a significant seroascencion (atleast a fourfold raise in reciprocal titers), according to the WHOrecommended standards. Probable cases were defined as clinical andepidemiological suspicion together with a unique serum with a MATreciprocal titer greater than 400 [Berlioz-arthaud A. et al, Trans R.Soc. Trop. Med. Hyg. 2007]. The panel of strains used for MAT wasadapted to the local epidemiology.

All sera used in this study were addressed to Institut Pasteur fordiagnostic purpose and originated from patients from New Caledonia,mainland France and the French West Indies (Martinique and Guadeloupe).They were stored at −20° C., selected according to case definitions,then tested blindly. To assess the sensitivity of the dipstick assay,only MAT positive sera from confirmed cases were used. The specificitywas assessed using MAT negative healthy or pathological sera from thereference laboratories of the Institut Pasteur in Nouméa (IPNC) orParis.

The IPNC and the NRC are reference diagnostic laboratories forleptospirosis. In New Caledonia, leptospirosis is a notifiable disease.The serum samples used in this study were selected from the IPNC and NRCcollections of sera issued from routine diagnostic activities and aspart of public health surveillance. This biobank of sera was declared tothe French Ministry of Research (DC-2010-1222, Collections number 1 and2). This study was part of a protocol approved by the Institut Pasteur(protocol # RBM2008-16) and the French Minister for Education & Research(protocol # AC-2007-44). All sera were tested as anonymous samples.

3.2. Dipstick Protocol

The positive control line was made of purified human IgM (MPBiomedicals) at 2 mg/mL. Both control and test lines were sprayed aslines onto nitrocellulose membranes. The crude formalin-treated bacteriapreparation was directly used as a fixed antigen for the test line. Goldparticles labelled with goat anti-human IgM (BBI InternationalBA.GAHM40/X) was used as the capture mobile phase to construct ourone-step vertical flow immunochromatography dipsticks, as describedbefore [Chanteau S. et al, PLoS Med, 2006].

Preliminary experiments determined a 1/400 dilution of sera in PhosphateBuffer Saline (PBS, pH 7.4) as suitable. Briefly, strips were introducedinto 200 μL diluted serum in 5 mL polystyrene tubes, for 15 minutes. Thestrips were then removed and placed on absorbent kitchen paper and readwithin 5 minutes. All results were recorded using a grading scale from 0(no visible trace on test band) to 3+(intensity of the test band equalto the intensity of the control band). The grading included a “weak”value for low but visible traces on the test band. Weaks, 1+, 2+ and 3+were then considered positive for further analysis and 0 was consideredas negative. All the strips were archived for further checking.

All analyses were run blindly: any person involved in one particularanalysis had no access to the results of the other tests results fromthe same serum. The sensitivity was evaluated using 187 confirmedleptospirosis cases sera with a MAT reciprocal titer 400. Thespecificity was assessed using 221 negative sera (142 from NewCaledonia, 79 from mainland France): 12 anti-Chikungunya virus IgMpositive sera, 58 anti-dengue virus IgM positive sera from all 4serotypes, 6 anti-hepatitis A virus total Ig positive sera, 7 rheumatoidfactor positive sera, 25 syphilis (TPHA and VDRL) positive sera, oneacute malaria serum and 112 sera from healthy blood donors. All these221 negative control sera have been tested using MAT and were allnegative (titer <100).

Possible false negative result due to high level of anti-Leptospira IgM(zone phenomenon) was controlled using two positive sera with 25,600 and51,200 MAT titers, respectively.

3.3. Stability Assays

The strips were stored at 4° C. in sealed aluminium foils, and the testwas performed at laboratory room temperature (20°-23° C.).

The predictive shelf life of the coated-strips was assessed by testingserial dilutions of a MAT positive serum (MAT reciprocal titer=800,pointing to serogroup Icterohaemorrhagiae) twice per week over a periodof 3 weeks exposure of the strips at 60° C. During this period, thepositive control serum was kept at 4° C. to avoid repeated freeze-thawcycles. This accelerated stability method is equivalent to two years ofactual storage time at 25° C. [Banoo et al, Nat. Rev. Microbiol. 2010].

3.4. Reproducibility and Repeatability

Several experiments were performed to evaluate the robustness of thedipstick assay (reproducibility and repeatability):

-   -   Simulation of tropical fields conditions by performing the tests        in parallel at laboratory room temperature and at 37° C. in an        incubator;    -   blind testing of a panel of four sera (3 positives and one        negative) by three different operators on three different days;    -   blind testing of one same serum 14 times using two different        batches of strips by the same operator.    -   blind reading of strips results by two technicians on 117 sera        (28 negative and 89 positive samples), and by three technicians        on 97 sera (16 negative and 81 positive samples).        3.5. Comparative Kinetics of MAT and the Dipstick Assay of the        Invention

The earliness of IgM seroconversion using MAT or the dipstick assay ofthe invention was assessed on serial sera (day 2 to day 18 after theonset of symptoms) from 17 confirmed cases, based on the date of onsetas declared by the patients.

The dipstick assay was also used on early sera from 99 PCR positiveconfirmed patients but still MAT negative (reciprocal titers=0, 100 or200).

150 MAT positive sera from probable cases, including 124 sera from theIPNC collection, and 26 from the French National Reference Centre, weretested using this dipstick assay.

3.6. Comparison with 3 Commercial Diagnostic Assays

To compare the newly developed RDT with currently available techniques,the performance of these assays was compared on identical sera from NewCaledonia. To assess the sensitivity, 72 MAT-positive sera fromconfirmed cases were randomly selected from the 118 New Caledoniancontrol sera. For the specificity, 72 negative controls wererandomly-selected, corresponding to 10 anti-Chikungunya virus IgMpositive sera, 30 healthy blood donors, 11 anti-dengue virus IgMpositive sera from all 4 serotypes, 6 anti-hepatitis A virus total Igpositive sera, 7 rheumatoid factor positive sera, 7 syphilis (TPHA andVDRL) positive sera, one acute malaria serum. The results using thedipstick assay of the invention were compared with those obtained usingtwo Elisa assays (Leptospira IgM ELISA, Panbio, Inverness Medical, QLDAustralia, and SERION ELISA classic Leptospira IgG/IgM, InstitutVirion/Serion GmbH, Germany) and one lateral flow IgMimmunochromatography assay (Leptocheck, Zephyr Biomedicals, India). TheSerion ELISA test was used together with the Rheumatoid Factor Absorbentas recommended by the manufacturer. All tests were made within a 5 dayperiod. For calculations, the “uncertain” results of ELISA wereconsidered as positive.

3.7. Statistical Analysis

The evaluation of the dipstick assay of the invention for theserodiagnosis of leptospirosis was performed according to the WHOTropical Diseases Research Diagnostics Evaluation Expert Panel for theevaluation of diagnostic tests for infectious diseases [Banoo et al,Nat. Rev. Microbiol. 2010].

Sensitivity (Se), specificity (Sp), positive and negative predictivevalues (PPV and NPV, respectively) of the dipstick assay werecalculated, using the reference MAT serology as the gold standard. Bothtests were conducted at the IPNC. The 95% confidence intervals (95% CI)were calculated using the Wilson method.

The likelihood ratios (LR) were also calculated. The positive LR(LR+=Se/[1−Sp]) indicates how many times a positive result is morelikely to be observed in specimens with the target disorder than inthose without the target disorder. The negative LR (LR−=[1−Se]/Sp)indicates how many times a negative result is more likely to be observedin specimens with the target disorder than in those without the targetdisorder. The test is more accurate the more LR differs from 1. LR+above 10 and LR− below 0.1 were considered convincing diagnosticevidence [Jaeschke R et al, JAMA 1994]. The 95% CIs were calculated forLR+ and LR− [Simel D L. J. CLin. Epidemiol. 1991].

The diagnostic odds ratio (DOR) measures of test performance bycombining the strengths of sensitivity and specificity, with theadvantage of accuracy as a single indicator. These characteristics lendthe DOR particularly useful for comparing tests whenever the balancebetween false negative and false positive rates is not of immediateimportance [Glas A S. et al, J. Clin. Epidemiol. 2003]. The DOR isdefined as the ratio of the odds of positive test results in specimenswith the target disorder relative to the odds of positive test resultsin specimens without the target disorder. It was calculated as follows:DOR=(Se/[1−Se])/([1−Sp]/Sp)

The DOR does not depend on prevalence and its value ranges from 0 toinfinity, with higher values indicating better discriminatory testperformance. The 95% CIs for DOR values were calculated [Armitage P. etal, Statistical methods in medical research, 1994].

3.8. Results of the disptick assay of the invention

a) Specificity and Sensitivity

Out of the 187 gold standard positive sera tested, 168 had a positivedipstick result. The putative serogroups of the 19 dipstick negativesera were: Icterohaemorrhagiae (n=12), Pyrogenes (n=3), Australis (n=2),Panama (n=1) and one could not be determined due to co-agglutination ofmultiple serogroups.

Out of the 221 MAT negative sera tested, 207 were had a negativedipstick result. All 14 dipstick positive sera were graded “weak” andoriginated from 9 healthy blood donors and five patients positive foranti-dengue virus IgM.

The sensitivity and specificity of the dipstick assay of the inventionwere therefore, respectively, Se=89.8% [95% CI, 84.7-93.4] and Sp=93.7%[95% CI, 89.65-96.2]. The Likelihood Ratios (LR) were thereforeLR+=14.18 [95% CI, 8.52-23.56] and LR−=0.11 [95%; 0.01-0.17]; and theDiagnostic Odds Ratio DOR of 130.74 [95% CI, 63.65-268.52].

The positive and negative predictive values according to prevalence arepresented in FIG. 1.

The absence of false negative due to zone phenomenon was demonstratedusing two positive sera with very high MAT reciprocal titers (25,600 and51,200) serially two-fold diluted (1/400 to 1/6,400).

b) Temperature Stability and Accelerated Aging Method for Shelf Life

The dipstick results of 10 MAT positive sera run at 37° C. wereidentical to those run at 25° C.

Serial two-fold dilutions (from 1/400 to 1/12,800) of one MAT positiveserum (titer 800) where tested twice a week for three weeks withdipstick exposed at 60° C. At day 1, the dipstick reciprocal titer was6,400, and remained the same till day 17. At day 21, the reciprocaltiter decreased to 3,200 (one dilution of the serum).

c) Reproducibility and Repeatability

One serum tested 14 times with strips from the two different batchesgave 14 similar results, including the grade.

Inter-readers variability was assessed by two independent operators on177 sera (28 negative and 149 positive) of which 157 sera were read bythree independent operators. These readings provided an excellentinter-operator agreement in all cases (>99%) but one weakly positivedipstick from a probable case was rated “weak” by two operators butnegative by the third one.

Inter-operator variability was also assessed using 4 sera (dipstickgraded from negative to 3+) blindly and independently tested at threedifferent days by three different operators. Two operators providedperfectly concordant grading results on all three tests, the third onegraded “weak” a negative serum once out of the three tests.

d) Comparative Kinetics of MAT and RDT

TABLE 2 MAT RDT Patient Leptospirosis diagnosis Putative serogroup Seratested * positive* positive* 1 blood PCR+ at D4 Icterohaemorrhagiae D4-6D6 — 2 blood PCR+ at D8 Icterohaemorrhagiae D8-9; D11-14 — D9 3 bloodPCR+ at D2 Icterohaemorrhagiae D2-6 — D5 4 blood PCR+ at D4Icterohaemorrhagiae D4; D6-12 — D6 5 blood PCR+ at D1 Pyrogenes D1-2;D4-5; D7 — D7 6 blood PCR+ at D4 Ballum D4-6 — D4 7 blood PCR+ at D6Icterohaemorrhagiae D6-8; D11  D11 8 blood PCR+ at D4Icterohaemorrhagiae D4-8 D7 9 Seroconversion D4-D7 IcterohaemorrhagiaeD4; D7; D9 D7 10 urine PCR+ at D5 Icterohaemorrhagiae D5-6 D5 11Seroascension D5-D9 Icterohaemorrhagiae D6, D9, D11 D6 12 blood PCR+ atD5 Icterohaemorrhagiae D5-8; D10; D12-13  D13 D5 13 urine PCR+ at D8Icterohaemorrhagiae D7-12; D17  D17 D7 14 blood PCR+ at D3Icterohaemorrhagiae D3-6 D6 D5 15 blood PCR+ at D4 IcterohaemorrhagiaeD3-7; D9-12 D5 D4 16 blood PCR+ at D7 Icterohaemorrhagiae D7-11; D13-17 D17 D7 17 blood PCR+ at D3 Icterohaemorrhagiae D3-6 D6 D3

Of 17 confirmed cases analysed (see Table 2):

-   -   one patient (number 1) seroconverted for MAT at day 6 (pointing        to Icterohaemorrhagiae) but remained negative for the dipstick        assay.    -   oppositely, 5 PCR confirmed patients (numbers 2-6) were MAT        negative whereas they were positive for the dipstick assay. For        one of these patients (number 6), PCR and dipstick assays were        both positive at day 4 after onset of symptoms.    -   five patients (numbers 7-11) were positive for MAT and the        dipstick assay on the same day (days 5-11 after the onset of        symptoms);    -   lastly, for 6 patients (numbers 12-17), the dipstick assay was        positive earlier than the MAT (day 3 to day 7). Out of these 6,        four (numbers 12, 15, 16 and 17) had a positive blood PCR and        dipstick results on the same day (on days 5, 4, 7 and 3        respectively).

Similarly, in 16 out of 99 early sera from confirmed patients from NewCaledonia, the dipstick assay was positive whereas the MAT was stillnegative (6 out of 62) or displayed low titers (titer 100 for 4 out of21; titer 200 for 6 out of 16).

Of 150 sera from probable cases of leptospirosis (unique sera with a MAT400), 109 gave a positive result using the dipstick assay of theinvention, corresponding to a concordance of 72.7% [65-79.1].

Out of these, 108 had a MAT>400, from which 81 (75% [66.1-82.2]) werepositive for the dipstick assay of the invention, while 63 had a MATtiter >800, from which 53 (84.1% [73.2-91.1]) were positive for thedipstick assay of the invention.

The use of 72 Gold Standard positive (MAT≧400 from confirmed cases) and72 negative (MAT<100) serum specimens selected randomly allowed acomparison of the dipstick assay of the invention with threecommercially available tests: two ELISA tests and one IgM lateral flowimmunochromatographic assay.

The results of these tests are detailed in Table 3.

TABLE 3 Dipstick Elisa Serion Elisa MAT 1/400 e Leptocheck (+RFabsorbant) Panbio Positive 58 Positive 53 Positive 59 Negative 5Negative 1 Positive 0 Positive 59 Negative 1 Positive 0 Positive 0Negative 0 Negative 0 Positive 72 Negative 0 Positive 0 Negative 0Positive 8 Positive 1 Positive 11 Negative 7 Negative 3 Positive 0Negative 13 Negative 3 Positive 0 Positive 0 Negative 2 Negative 0Negative 2 Positive 0 Negative 2 Sensitivity 81.9% 97.2% 91.7%  75%[71.5-89.1] [90.4-99.2] [83-86.1] [63.9-83.6] Negative 72 Negative 69Negative 38 Negative 59 Negative 72 Specificity 95.8% 52.8% 81.9% 100%[88.4-98.6] [41.4-63.9] [71.5-89.1] [94.9-100] LR+ 19.7 [6.5- 2.1 [1.6-5.1 [3.1-8.3] NA 59.9] 2.6] LR− 0.2 [0.1-0.3] 0.05 [0.01- 0.1 [0.05-0.2]0.25 [0.17- 0.21] 0.37] DOR 104.4 [28.4- 39.1 [8.9- 49.9 [17.8- NA 384]171.8] 139.7] NA: Not Applicable * LR+: Positive Likelihood Ratio—[95%CI] † LR−: Negative Likelihood Ratio—[95% CI] ‡ DOR: Diagnostic OddsRatio—[95% CI]

Sensitivity (%), number of positive rapid diagnostic test among patientswith serological evidence (MAT) of leptospirosis (n=72)−[95% CI].

Specificity (%), negative rapid diagnostic test among serum samples frompatients with no serological evidence (MAT) of leptospirosis (n=72)−[95%CI].

The IgM ELISA from Panbio had 100% specificity on these specimenstogether with the lowest sensitivity (75%). This 100% specificity doesnot allow the calculation of a Diagnostic Odds Ratio (DOR) that wouldhowever be very high. The ELISA test from Serion had both a goodsensitivity (91.7%) and a good specificity (81.9%), therefore showing agood DOR of 49.9. Another rapid diagnostic test, namely Leptocheck (fromZephyr) had a very good sensitivity (91.2%) but a quite low specificity(52.8%), giving a DOR of 39.1. The Dipstick assay of the inventiondisplayed a very good specificity (95.8%) and a good sensitivity (81.9%)and had therefore a very good DOR of 104.4. The corresponding curves ofpredictive values according to the prevalence of the two IgM rapid testson these specimens are compared in the FIG. 2.

When considering the need of RDT for bedside diagnosis, the comparisonof the Dipstick assay of the invention with assays that are commerciallyavailable shows that the dipstick assay of the invention has a lowersensitivity (81.9% versus 97.2%) but a much higher specificity (95.8%versus 52.8%) and therefore a better Diagnostic Odds Ratio (104.4 versus39.1). This better performance is also shown by the comparison of thecurves of their predictive values according to prevalence (FIG. 2).

Importantly, only sera from confirmed leptospirosis cases were used forthis evaluation. Therefore, the positive samples for the evaluation ofsensitivity were both Gold Standard positive (a MAT reciprocal titer ofat least 400) and from confirmed leptospirosis cases (either a positivePCR or a seroconversion from nil to ≧400 or a ≧4-fold rise in MATreciprocal titers in paired sera). Additionally, all negative sera forspecificity evaluation were tested blindly using the reference MAT andwere only considered as true negatives if the MAT reciprocal titer wasbelow 100. These latter originated from both healthy volunteers and aselection of patients with pathologic conditions of relevance in endemiccountries. Using this clearly defined case definition, the sensitivityand specificity of the dipstick assay of the invention were 89.8% and93.7% respectively. These results compare and are slightly better thanthe ones reported by Smits et al. who reported a 85.8% sensitivity and a93.6% specificity with another Dip Stick assay (Smits H L. Clin. Diagn.Lab. Immunol. 2001). To increase the statistical power of thisevaluation, were included serum samples as old as 3 years and 3 monthsfrom New Caledonia, stored frozen at −20° C. It is well recognized thatthe long term storage of serum specimens at −20° C. and theirfreeze/thawings may result in a drop of IgM titers. Actually, thesensitivity was higher in sera stored for less than two years than insera stored for more than two years (90.6% versus 81.5%). This may haveresulted in a slight under-estimation of the sensitivity of the Dipstickassay of the invention.

Importantly, the dipstick assay of the invention reacts with antibodiesto at least serogroups Australis, Autumnalis, Ballum, Bataviae,Canicola, Cynopteri, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae,Panama, Pomona, Pyrogenes, Sejroe and Tarassovi, indicating that theassay reacts broadly with antibodies mounted against Leptospira strainscirculating worldwide.

These results demonstrate that the diagnostic test of the invention isuseful in endemic contexts, especially in low and middle-incomecountries. Actually, most of the leptospirosis burden occurs in theback-country with delayed access to the reference laboratory. Inepidemics situations, especially during post-disaster periods like inthe Philippines in 2009, reference diagnostic tests are seldom if everavailable. Therefore, a diagnosis test with good diagnostic performanceswould also be particularly useful. The use of the dipstick assay of theinvention as an initial screen for leptospiral infections would allowfacilitating the difficult differential diagnosis of leptospirosis.

The invention claimed is:
 1. An in vitro method for diagnosing aLeptospira infection in a biological sample of a subject, comprising:providing a biological sample of a subject; contacting said sample withheat and chemical inactivated bacterial cells of Leptospira faineiserovar Hurstbridge, or an antigenic fraction of said heat and chemicalinactivated bacterial cells, wherein said heat and chemical inactivatedbacterial cells or antigenic fraction thereof are immobilized on a solidsupport; and assaying for binding of antibodies in said biologicalsample with said heat inactivated bacterial cells or antigenic fractionthereof immobilized on said solid support; wherein said heat andchemical inactivated bacterial cells or antigenic fraction thereofimmobilized on said solid support react with antibodies againstLeptospira serogroups Australis, Autumnalis, Ballum, Bataviae, Canicola,Cynopteri, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Panama,Pomona, Pyrogenes, Sejroe, and Tarassovi.
 2. The method of claim 1,wherein binding of antibodies in said biological sample with said heatand chemical inactivated bacterial cells or antigenic fraction thereofimmobilized on said solid support is detected.
 3. The method of claim 1,wherein binding of antibodies in said biological sample with said heatand chemical inactivated bacterial cells or antigenic fraction thereofimmobilized on said solid support is not detected.
 4. The method ofclaim 1, wherein said antigenic fraction is selected fromlipopolysaccharide (LPS), cytoplasmic proteins, secreted proteins, andenvelope membrane proteins.
 5. The method of claim 4, wherein saidantigenic fraction is LPS.
 6. The method of claim 1, wherein saidleptospirosis infection is due to bacteria belonging to at least oneserogroup selected from the group consisting of: serogroup Australis,Autumnalis, Ballum, Bataviae, Canicola, Cynopteri, Grippotyphosa,Hebdomadis, Icterohaemorrhagiae, Panama, Pomona, Pyrogenes, Sejroe, andTarassovi.
 7. The method of claim 1, wherein the method is an ELISAassay.
 8. The method of claim 1, wherein the method is a dipstick assay.